In the two-hybrid system of Fields and Song(1) described in U.S. Pat. No. 5,283,173 two chimeric genes which encode hybrid proteins are used to test the interaction between a known protein and protein of interest. The first chimeric gene codes for a known protein, often called the bait protein, fused to a DNA-binding domain. The second chimeric gene codes for a protein of interest fused to the transcriptional activation domain. Additionally, the protein of interest may not be known and could be derived for example from a cDNA library. In a suitable host cell such as yeast, if the protein of interest and the bait protein do interact they bring into proximity the DNA-binding and transcriptional activation domains. This proximity is sufficient to cause transcription of a marker gene placed under the control of a promoter containing a binding site for the DNA-binding domain.
Yeast genetic systems have also been used in methods for defining DNA-binding domains of proteins. One such method (2) uses a chimeric protein containing a transcriptional activation domain together with two DNA-binding domains, each capable of binding to a different reporter gene. One of the DNA-binding domains is mutated to analyse its DNA-binding properties.
In the two-hybrid system, once a specific cDNA-encoded protein or known protein of interest has been shown to give rise to activation of the marker gene, it is important to show that this is indeed due to an interaction between the bait protein and the protein of interest and not due to a "false positive" interaction (3). At least four classes of false positives may occur in two-hybrid systems where a protein of interest, which may be encoded by a cDNA library, is fused to the activation domain:
1. A DNA-binding domain might be cloned into the activator hybrid which would activate transcription independent of the DNA-binding hybrid, by binding: PA1 2. Protein in the activator hybrid might bind to the DNA-binding domain of the bait hybrid; PA1 3. Protein in the activator hybrid might bind to the novel junction formed between the protein and an epitope tag or tags encoded by the bait hybrid vector, or to the epitope tag(s) itself; PA1 4. Protein in the bait hybrid might bind to the novel junction formed between the protein of interest and an eptiope tag or tags encoded by the activator hybrid vector. PA1 a) providing a host cell; PA1 b) providing a first chimeric gene that is capable of being expressed in the host cell, the first chimeric gene comprising a DNA sequence that encodes a first hybrid protein, the first hybrid protein comprising: PA1 c) providing a second chimeric gene that is capable of being expressed in the host cell, the second chimeric gene comprising a DNA sequence that encodes a second hybrid protein, the second hybrid protein comprising: PA1 d) providing a control chimeric gene that is capable of being expressed in the host cell, the control chimeric gene comprising a DNA sequence that encodes a control hybrid protein, the control hybrid protein comprising: PA1 e) introducing the first chimeric gene and the second chimeric gene into the host cell and subjecting the host cell to conditions under which the first and second hybrid proteins are expressed in sufficient quantity for the first detectable signal to be produced; PA1 f) introducing the control chimeric gene into the host cell in the presence of the first and second chimeric genes and subjecting the host cell to conditions under which the control hybrid protein is expressed in sufficient quantity for the second detectable signal to be produced; PA1 g) determining whether either or both of the detectable signals are produced. PA1 b) (ii) the first detection domain comprises a first DNA-binding domain which recognises a DNA binding site on the first reporter gene; PA1 c) (ii) the second detection domain comprises a transcriptional activation domain; and PA1 d) (ii) the control detection domain comprises a second DNA-binding domain which recognises a DNA binding site on the second reporter gene; PA1 a) providing a host cell containing a first reporter gene under the control of a first promoter which is activated by a transcriptional activation domain; PA1 b) providing a first chimeric gene that is capable of being expressed in the host cell, the first chimeric gene comprising a DNA sequence that encodes a first hybrid protein, the first hybrid protein comprising: PA1 c) providing a second chimeric gene capable of being expressed in the host cell, the second chimeric gene comprising a DNA sequence that encodes a second hybrid protein, the second hybrid protein comprising: PA1 d) introducing the first chimeric gene and the second chimeric gene into the host cell; PA1 e) subjecting the host cell to conditions under which the first hybrid protein and the second hybrid protein are expressed in sufficient quantity for the detectable gene to be activated; PA1 f) determining whether the detectable gene has been expressed; PA1 g) further providing a second reporter gene under the control of a second promoter which is activated by a transcriptional activation domain, said second promoter having a different DNA binding site to the first promoter and preferably also lacking as far as possible any DNA binding motif in common with the first promoter in the basal portion of the promoter; PA1 h) further providing a control chimeric gene that is capable of being expressed in the host cell, the control chimeric gene comprising a DNA sequence that encodes a control hybrid protein, the control hybrid protein comprising: PA1 (i) introducing the control chimeric gene and the second reporter gene into the host cell; PA1 (i) subjecting the host cell to conditions under which the control hybrid protein and the second hybrid protein are expressed in sufficient quantity for the second reporter gene to be activated; and PA1 (k) determining whether the second reporter gene has been expressed to confirm whether the first and second test proteins interact. PA1 (a) two different bait vectors capable of expressing two different hybrid proteins comprising different DNA-binding domains and a first test protein, each vector comprising a nucleic acid sequence which encodes one of the DNA-binding domains and an insertion site for a nucleic acid sequence encoding the first test protein; or PA1 (b) a single bait vector capable of expressing a dual bait hybrid protein comprising two different DNA-binding domains and a first test protein the vector comprising nucleic acid sequences which encode the DNA-binding domains and an insertion site for a nucleic acid sequence encoding the first test protein; and PA1 (c) an activator vector capable of expressing an activator hybrid protein comprising a transcriptional activation domain and a second test protein, the vector comprising a nucleic acid sequence which encodes the activation domain and an insertion site for a nucleic acid sequence encoding the second test proteins; and PA1 (d) two reporter genes capable of producing different detectable signals, each reporter gene having a DNA binding site for one of the DNA-binding domains encoded by the bait vector or vectors.
a) to the DNA binding site or PA2 b) to the basal portion of the promoter; PA2 (i) a first test protein that is to be tested for interaction with a second test protein; and PA2 (ii) a first detection domain; PA2 (i) a second test protein that is to be tested for interaction with the first test protein; and PA2 (ii) a second detection domain; PA2 (i) the first test protein; and PA2 (ii) a control detection domain; PA2 wherein when the first detection domain and the second detection domain are brought into proximity to one another by an interaction between the first and second hybrid proteins, a first detectable signal is produced, and when the second detection domain and the control detection domain are brought into proximity to one another by an interaction between the second hybrid protein and the control hybrid protein a second detectable signal is produced which is distinguishable from the first detectable signal; PA2 (i) a first test protein that is to be tested for interaction with a second test protein; PA2 (ii) a DNA-binding domain that recognises a binding site on the first promoter; PA2 (i) a second test protein that is to be tested for interaction with the first test protein; and PA2 (ii) the transcriptional activation domain; PA2 (i) the first test protein; PA2 (ii) a DNA-binding domain that recognises a binding site on the second reporter gene:
A number of strategies have been previously described which remove some of the above classes of false positives (3). Three such strategies are as follows:
1. The use of two marker genes (3). One of these genes usually expresses a selectable marker (eg. HIS3) and the other one a measurable marker activity (eg. lacZ) and the reporter gene promoters are usually different. This allows the removal of those proteins in the first class of false positive where a non-specific interaction occurs between the protein and the promoter of the marker gene, since non-specific interaction with both of the marker gene promoters is less likely to occur.
2. The use of curing to remove the bait hybrid protein (4). This strategy removes the bait hybrid plasmid, so that only the activator hybrid expressing the protein of interest is present in the cell. If the marker gene expression remains high, then this shows that the activation is due to spurious promoter activation by the protein of interest hybrid protein, rather than via binding through the bait protein hybrid.
3. The use of an unrelated bait protein (3). This is to ensure that the protein interacts with the bait protein of interest and no other part of the bait hybrid protein.
A review by Allen et al 1995 (5) describes strategies for the removal of false positives. One of these, elaborated on in Harper et al 1993 (6), involves curing out the bait hybrid and using unrelated bait proteins fused to the DNA-binding domain of the original bait protein, to detect false positives.
Another strategy is described by Le Douarin et al 1995 (7). When an apparent positive interaction is detected, the bait hybrid protein is removed by curing and a bait hybrid containing a different DNA-binding domain is introduced. False positives are indicated if there is activation of a reporter construct to which the different DNA-binding domain binds.
Strategies which have previously been applied to the problem of false positives in two-hybrid systems generally remove only some of the above classes of false positives. Known strategies can in principle be used consecutively to remove all false positives, but this will entail lengthy and complicated procedures. The previously used strategies which do remove several classes of false positives require lengthy and labour intensive screening steps after the initial screen. No strategy has been devised which would be able to remove all of these false positives at the initial screening step. Furthermore, there has so far been no strategy for removing false positives in the screening of two libraries against each other in a search for pairs of interacting proteins. Clearly, there is a need for methods of effectively eliminating false positives in these systems.